Masanori P. Takahashi

Expanded CUG Repeats Trigger Aberrant Splicing of ClC-1 Chloride Channel Pre-mRNA and Hyperexcitability of Skeletal Muscle in Myotonic Dystrophy

In myotonic dystrophy (dystrophia myotonica, DM), expression of RNAs that contain expanded CUG or CCUG repeats is associated with degeneration and repetitive action potentials (myotonia) in skeletal muscle. Using skeletal muscle from a transgenic mouse model of DM, we show that expression of expanded CUG repeats reduces the transmembrane chloride conductance to levels well below those expected to cause myotonia. The expanded CUG repeats trigger aberrant splicing of pre-mRNA for ClC-1, the main chloride channel in muscle, resulting in loss of ClC-1 protein from the surface membrane. We also have identified a similar defect in ClC-1 splicing and expression in two types of human DM. We propose that a transdominant effect of mutant RNA on RNA processing leads to chloride channelopathy and membrane hyperexcitability in DM.

Misregulation of miR-1 processing is associated with heart defects in myotonic dystrophy.

Myotonic dystrophy is an RNA gain-of-function disease caused by expanded CUG or CCUG repeats, which sequester the RNA binding protein MBNL1. Here we describe a newly discovered function for MBNL1 as a regulator of pre-miR-1 biogenesis and find that miR-1 processing is altered in heart samples from people with myotonic dystrophy. MBNL1 binds to a UGC motif located within the loop of pre-miR-1 and competes for the binding of LIN28, which promotes pre-miR-1 uridylation by ZCCHC11 (TUT4) and blocks Dicer processing. As a consequence of miR-1 loss, expression of GJA1 (connexin 43) and CACNA1C (Cav1.2), which are targets of miR-1, is increased in both DM1- and DM2-affected hearts. CACNA1C and GJA1 encode the main calcium- and gap-junction channels in heart, respectively, and we propose that their misregulation may contribute to the cardiac dysfunctions observed in affected persons.

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate–binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.

Muscleblind-like 2-Mediated Alternative Splicing in the Developing Brain and Dysregulation in Myotonic Dystrophy

The RNA-mediated disease model for myotonic dystrophy (DM) proposes that microsatellite C(C)TG expansions express toxic RNAs that disrupt splicing regulation by altering MBNL1 and CELF1 activities. While this model explains DM manifestations in muscle, less is known about the effects of C(C)UG expression on the brain. Here, we report that Mbnl2 knockout mice develop several DM-associated central nervous system (CNS) features including abnormal REM sleep propensity and deficits in spatial memory. Mbnl2 is prominently expressed in the hippocampus and Mbnl2 knockouts show a decrease in NMDA receptor (NMDAR) synaptic transmission and impaired hippocampal synaptic plasticity. While Mbnl2 loss did not significantly alter target transcript levels in the hippocampus, misregulated splicing of hundreds of exons was detected using splicing microarrays, RNA-seq, and HITS-CLIP. Importantly, the majority of the Mbnl2-regulated exons examined were similarly misregulated in DM. We propose that major pathological features of the DM brain result from disruption of the MBNL2-mediated developmental splicing program.

Intracellular calcium increase induced by GABA in visual cortex of fetal and neonatal rats and its disappearance with development.

To address the question of whether gamma-aminobutyric acid (GABA) induces a change in the concentration of Ca2+ in neurons of the developing visual cortex, and if so, to elucidate a developmental profile of such a GABA-induced change, we measured intracellular Ca2+ signals using microscopic fluorometry in visual cortical slices loaded with rhod-2. The slices were prepared from rat fetuses of embryonic day 18 (E18) and rat pups of postnatal days 0-30 (P0-P30). Application of GABA through the perfusate at 100 microM induced a marked rise in intracellular Ca2+ signals in the cortical plate and subplate at E18 and P0-P2. After P5 the GABA-induced rise in Ca2+ dramatically reduced, and at P20 and thereafter it became undetectable. At E18 and P0-P2 an agonist for GABAA receptor, muscimol, induced a Ca2+ rise in the same way as did GABA, while a GABAB receptor agonist, baclofen, did not induce any significant rise in Ca2+ signals. Also, a GABAA receptor antagonist, bicuculline, blocked the GABA-induced rise in Ca2+ signals. These results indicate that the Ca2+ rise is triggered by activation of GABAA receptors. The application of Ni2+ at a concentration high enough to block all types of voltage-dependent CA2+ channels prevented the Ca2+ signals from increasing in response to GABA application, suggesting that Ca2+ may be influxed through such channels following depolarization evoked by GABA.

Enhanced Slow Inactivation by V445M: A Sodium Channel Mutation Associated with Myotonia

Over 20 different missense mutations in the alpha subunit of the adult skeletal muscle Na channel have been identified in families with either myotonia (muscle stiffness) or periodic paralysis, or both. The V445M mutation was recently found in a family with myotonia but no weakness. This mutation in transmembrane segment IS6 is novel because no other disease-associated mutations are in domain I. Na currents were recorded from V445M and wild-type channels transiently expressed in human embryonic kidney cells. In common with other myotonic mutants studied to date, fast gating behavior was altered by V445M in a manner predicted to increase excitability: an impairment of fast inactivation increased the persistent Na current at 10 ms and activation had a hyperpolarized shift (4 mV). In contrast, slow inactivation was enhanced by V445M due to both a slower recovery (10 mV left shift in beta(V)) and an accelerated entry rate (1.6-fold). Our results provide additional evidence that IS6 is crucial for slow inactivation and show that enhanced slow inactivation cannot prevent myotonia, whereas previous studies have shown that disrupted slow inactivation predisposes to episodic paralysis.

Induction of LTD but not LTP through metabotropic glutamate receptors in visual cortex.

Long-term potentiation (LTP) and long-term depression (LTD), often used as essential components in synaptic models for learning, memory and forgetting, can be produced in cortical tissue by repetitive activation of neural pathways under different stimulus conditions. The involvement of metabotropic glutamate receptors (mGluRs) has been postulated to be necessary for the establishment of either or both forms of synaptic plasticity in hippocampus. The recent introduction of a specific antagonist for mGluRs, (+/-)-alpha-methyl-4-carboxyphenylglycine, prompted the investigation of the respective involvement of this receptor population in the induction of LTP and LTD in visual cortex of the rat in vitro. The results suggest the critical involvement of mGluRs in producing LTD but not LTP.

Muscleblind-Like 1 Knockout Mice Reveal Novel Splicing Defects in the Myotonic Dystrophy Brain

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTGexp) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA LR transgenic mouse model which expresses a pathogenic range CTGexp. In the present study, we addressed the possibility that MBNL1 sequestration by CUGexp RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1 ΔE3/ΔE3 knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1 ΔE3/ΔE3 knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1 ΔE3/ΔE3 brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUGexp RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.

Endoplasmic reticulum stress in myotonic dystrophy type 1 muscle

In myotonic dystrophy type 1 (DM1), alternative splicing of ryanodine receptor 1 (RyR1) and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) genes has been reported. These proteins are essential for maintaining intracellular Ca2+ in skeletal muscle. To clarify involvement of endoplasmic reticulum (ER) stress in DM1 muscles, we examined the activation of ER stress-related proteins by immunohistochemistry, western blot analysis and RT-PCR. In four of five DM1 muscle biopsies, except for a muscle biopsy from a patient with the shortest CTG expansion and no myotonia, increased expression of GRP78 and calnexin, and phosphorylation of PERK and eIF-2α were revealed in fibers with sarcoplasmic masses and in highly atrophic fibers with pyknotic nuclear clumps. Caspase-3 and -7 were also expressed in these fibers. Increased expression of GRP78 in these DM1 muscles was confirmed by western blot analysis. GRP78 mRNA and spliced isoform of XBP1 mRNA were also increased in DM1 muscle biopsies. Furthermore, we demonstrated increased expression of GRP78 in highly atrophic fibers with pyknotic nuclear clumps in all three muscle biopsies from neurogenic muscular atrophies. However, five muscle biopsies from central core disease presumably with disturbed intracellular Ca2+ homeostasis and a muscle biopsy from paramyotonia congenita with myotonia showed no activation of these proteins. Taken together, ER stress is involved in muscle wasting in DM1. However, it seems to be evoked not only by disrupted intracellular Ca2+ homeostasis.

Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.